EP1565749A2 - Methods of screening for compounds that inhibit the biosynthesis of gpi in malaria parasites - Google Patents
Methods of screening for compounds that inhibit the biosynthesis of gpi in malaria parasitesInfo
- Publication number
- EP1565749A2 EP1565749A2 EP03774152A EP03774152A EP1565749A2 EP 1565749 A2 EP1565749 A2 EP 1565749A2 EP 03774152 A EP03774152 A EP 03774152A EP 03774152 A EP03774152 A EP 03774152A EP 1565749 A2 EP1565749 A2 EP 1565749A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- dna
- compound
- gpi
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/445—Plasmodium
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to methods of screening for compounds that inhibit the biosynthesis of GPI in malaria parasites.
- Malaria is the most common infectious human disease caused by parasitic protozoans. The disease is caused by infection with malaria parasites and is mediated by the mosquito, Anopheles gambiae. Every year there are estimated 500 million cases of malaria infection, including more than two million fatal cases (Gardner, et al . , Nature 419:498-511, 2003) . At present 40% of the world's population lives in malaria-epidemic areas, where it is said that one in every three infants dies from malaria.
- Glycosylphosphatidylinositol is known to play a key role in the growth and infectivity of parasites, including malaria parasites.
- GPI-anchored proteins include proteins such as MSP-1 that function when the parasites invade red blood cells. GPI proteins act as parasitic antigens and initiate an immune response in the host. Thus, they have long been the subject of research aimed at vaccine development.
- GPI not only functions as an anchor to tether proteins to the cell membrane, but is also an abundant glycolipid component of malaria parasite cell membranes. Recent studies have revealed that GPI is toxic and causes lethal symptoms. GPI induces the expression of inflammatory cytokines such as TNF- ⁇ , and of adhesion molecules. As a result, infected red blood cells adhere to capillaries, obstructing vessels (sequestration) , brain blood vessels in particular. This induces further inflammatory reactions that are believed to lead to encephalopathy. Very recently, Schofield et al .
- An objective of the present invention is to provide antimalarial drugs that inhibit the biosynthesis of GPI. More specifically, the present invention provides the malaria parasite DNA that encodes the GWTl protein, which is a protein involved in the biosynthesis of GPI (GPI synthase) . The present invention also provides a method of using this DNA in methods of screening for antimalarial drugs. The present invention also provides degenerate mutant DNAs of the DNA that encodes the malaria parasite GPI biosynthesis protein. These degenerate mutant DNAs have a lower AT content than the original DNA. The present invention also provides a method of using the degenerate mutant DNAs in methods of screening for antimalarial drugs.
- the GWTl gene was originally found to encode a fungal GPI-anchored protein synthase (WO 02/04626) , and is conserved in organisms ranging from yeasts to humans.
- the present inventors confirmed that GWTl homologues (PfGWTl for P. falciparum GWTl; PyGWTl for P. yoelii yoelii GWTl) are included in the entire genomic sequences of Plasmodium falciparum (P. falciparum) and Plasmodium yoelii yoelii (P. yoelii yoelii) (Gardner, et al .
- the GWTl gene product acts as a GlcN-PI acyltransferase in the GPI biosynthesis pathway.
- the PfGWTl gene product is expected to have similar activity, and thus compounds that inhibit this activity can be promising antimalarial drugs.
- WO 02/04626 the present inventors disclosed a group of compounds that inhibit the activity of the fungal GWTl gene product.
- Compounds inhibiting the activity of the PfGWTl gene product were expected to be antimalarial drugs.
- the present inventors succeeded in isolating a region thought to be almost the full length of the PfGWTl.
- antimalarial drugs can be screened through binding assays, glucosaminyl (acyl) phosphatidylinositol (PI-GlcN) acyltransferase assays, or using GPI-anchored protein detection systems. Compounds obtained from such screenings can be promising antimalarial drugs.
- degenerate mutant DNAs (degenerate mutants of the DNA that encodes the malaria parasite GPI biosynthesis protein) having a lower AT content than the original DNA, complement the phenotype of the GWTl gene-deficient fungus.
- degenerate mutant DNAs degenerate mutants of the DNA that encodes the malaria parasite GPI biosynthesis protein
- the present inventors revealed that degenerate mutant DNAs (degenerate mutants of the DNA that encodes the malaria parasite GPI biosynthesis protein) having a lower AT content than the original DNA, complement the phenotype of the GWTl gene-deficient fungus.
- the present invention provides the following [1] to [25] :
- an antimalarial drug which comprises as an active ingredient a compound that inhibits the activity of the protein according to [2];
- the antimalarial drug according to [5] wherein the compound that inhibits the activity of the protein according to [2] is at least one selected from the group consisting of the following compounds (1) to (5) :
- step (3) selecting a test sample that decreases the amount of the GPI-anchored protein transported to the cell wall, as determined in step (2) ;
- [11] a method for treating malaria, which comprises administering a compound that inhibits the activity of the protein according to [2];
- [17] a vector in which a DNA according to any one of [13] to [16] is inserted;
- [21] a method for producing a protein encoded by a DNA according to any one of [13] to [16], which comprises the steps of culturing the transformant according to any one of [18] to [20] , and recovering the expressed protein from the transformant or the culture supernatant;
- step (3) selecting a test sample that decreases the amount of the GPI-anchored protein transported to the cell wall, as determined in step (2);
- the DNA encoding the GWTl protein of Plasmodium fal ciparum (PfGWTl) was isolated for the first time in the present invention.
- the nucleotide sequence of the DNA encoding the PfGWTl protein is shown in SEQ ID NO: 1, and the amino acid sequence of the PfGWTl protein is set forth in SEQ ID NO: 2.
- the nucleotide sequence of the DNA encoding the GWTl protein of Plasmodium vivax (PvGWTl) is shown in SEQ ID NO: 3, and the amino acid sequence of the PvGWTl protein is set forth in SEQ ID NO: 4.
- the GWTl protein is involved in the biosynthesis of glycosylphosphatidylinositol (GPI) , which is essential for the growth and infectivity of malaria parasites.
- GPI glycosylphosphatidylinositol
- compounds that inhibit the activity of the malaria parasite GWTl protein can be used as antimalarial drugs.
- antimalarial drugs can be screened using this malaria parasite GWTl protein.
- the present invention provides DNAs encoding the malaria parasite GWTl protein.
- DNAs include DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4, and DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
- the present invention also provides DNAs encoding proteins that are functionally equivalent to the protein comprising the amino acid sequence of SEQ ID NO: 2 or 4.
- the expression “functionally equivalent” refers to having biological properties equivalent to those of the protein of interest, comprising the amino acid sequence of SEQ ID NO: 2 or 4 (the PfGWTl or PvGWTl proteins) .
- the biological properties of the PfGWTl and PvGWTl proteins include GlcN-PI acyltransferase activity.
- the GlcN-PI acyltransferase activity can be measured by the method reported by Costello and Orlean (J. Biol. Chem. (1992) 267:8599-8603), or Franzot and Doering (Biochem. J. (1999) 340:25-32).
- DNAs encoding proteins functionally equivalent to the protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 include: DNAs that hybridize under stringent conditions to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and DNA encoding a protein which comprises the amino acid sequence of SEQ ID NO: 2 or 4, in which one or more amino acids have been added, deleted, substituted, and/or inserted.
- the DNAs of the present invention can be isolated by methods well known to those skilled in the art. Examples of such methods include the use of hybridization (Southern E.M., J. Mol. Biol. 98: 503-517, 1975) and the polymerase chain reaction (PCR) (Saiki R.K. et al . , Science 230: 1350-1354, 1985; Saiki R.K. et al . , Science 239: 487-491, 1988) .
- hybridization Southern E.M., J. Mol. Biol. 98: 503-517, 1975
- PCR polymerase chain reaction
- DNAs that can be isolated by hybridization or PCR techniques, and that hybridize with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, are also comprised in the DNAs of the present invention.
- Such DNAs include DNA encoding a malaria parasite homologue of the protein comprising the amino acid sequence of SEQ ID NO: 2 or 4.
- the malaria parasite homologue includes those of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, which comprise the amino acid sequence of SEQ ID NO: 2 or 4.
- a DNA described above is isolated using hybridization reactions under stringent hybridization conditions.
- stringent hybridization conditions refers to, for example, hybridization in 4x SSC at 65°C followed by washing in 0. lx SSC at 65 °C for one hour.
- Alternative stringent conditions are hybridization in 4x SSC containing 50% formamide at 42 °C.
- the degree of identity at the amino acid sequence level or nucleotide sequence level can be determined using the BLAST algorithm of Karlin and Altschul (Karlin S. and Altschul S.F, Proc. Natl. Acad. Sci. USA.87: 2264-2268, 1990; Karlin S. and Altschul S . F, Proc. Natl. Acad. Sci. USA.90: 5873-5877, 1993) .
- BLAST algorithm-based programs called BLASTN and BLASTX, have been developed (Altschul S.F. et al . , J. Mol. Biol.215: 403, 1990) .
- nucleotide sequence is analyzed using BLASTN
- amino acid sequence is analyzed using BLASTX
- the default parameters for each program are used. Specific procedures for such analysis are known (please see the web site of the National Institute of Biotechnology Information http : //www. ncbi . nlm. nih . gov) .
- DNAs of the present invention comprise genomic DNAs, cDNAs, and chemically synthesized DNAs .
- a Genomic DNA or cDNA can be prepared according to conventional methods known to those skilled in the art.
- a genomic DNA can be prepared as follows: (i) extracting a genomic DNA from malaria parasites; (ii) constructing a genomic library (using, for example, a plasmid, phage, cosmid, BAC, or PAC, as a vector) ; (iii) spreading the library; and then (iv) conducting colony hybridization or plaque hybridization using probes prepared based on a DNA which encodes the malaria parasite GWTl protein of the present invention (e.g., SEQ ID NO: 1 or 3).
- genomic DNA can be prepared by PCR, using primers specific to a DNA which encodes the malaria parasite GWTl protein of the present invention (e.g., SEQ ID NO: 1 or 3) .
- cDNA can be prepared, for example, as follows: (i) synthesizing cDNA based on mRNA extracted from malaria parasites; (ii) constructing a cDNA library by inserting the synthesized cDNA into vectors such as ⁇ ZAP; (iii) spreading the cDNA library; and (iv) conducting colony hybridization or plaque hybridization as described above.
- the cDNA can also be prepared using PCR.
- the present invention also provides DNAs encoding proteins structurally similar to the protein comprising the amino acid sequence of SEQ ID NO: 2 or 4.
- DNAs include those which comprise nucleotide sequences encoding proteins comprising amino acid sequences in which one or more amino acid residues are substituted, deleted, inserted, and/or added.
- the percentage of mutated amino acids is typically 10% or less, preferably 5% or less, and more preferably 1% or less of the total amino acid residues.
- the number of mutated amino acids is usually 30 amino acids or less, preferably 15 amino acids or less, more preferably five amino acids or less, still more preferably three amino acids or less, even more preferably two amino acids or less.
- the mutant amino acid residue be one that retains the properties of the side-chain after its mutation (a process known as conservative amino acid substitution) .
- amino acid side chain properties are hydrophobicity (A, I, L, M, F, P, W, Y, V) and hydrophilicity (R, D, N, C, E, Q, G, H, K, S, T) .
- Side chains include: aliphatic side-chains (G, A, V, L, I, P) ; side chains containing an hydroxyl group (S, T, Y) ; side chains containing a sulfur atom (C, M) ; side chains containing a carboxylic acid and an amide
- a fusion protein comprising the malaria parasite GWTl protein is an example of a protein to which one or more amino acids residues have been added. Fusion proteins can be made by techniques well known to a person skilled in the art. For example, and without limitation to this particular technique, the DNA encoding the malaria parasite GWTl protein of the present invention can be combined with DNA encoding another peptide or protein such that their reading frames match. A protein of the present invention can form a fusion protein with a number of known peptides. Such peptides include FLAG (Hopp, T. P. et al .
- GST glutathione-S-transferase
- MBP maltose-binding protein
- the present invention provides vectors containing the DNAs of the present invention, transformants retaining the DNAs or vectors of the present invention, and methods for producing proteins of the present invention which utilize these transformants.
- a vector of the present invention is not limited so long as the DNA inserted into the vector is stably retained.
- pBluescript® vector (Stratagene) is preferable as a cloning vector when using E. coli as a host.
- An expression vector is particularly useful when using a vector to produce a protein of the present invention.
- the expression vector is not specifically limited, so long as it expresses proteins in vitro, in E. coli, in cultured cells, and in vivo.
- Preferable examples of expression vectors include the pBEST vector (Promega Corporation) for in vitro expression, the pET vector (Novagen) for expression in E. coli, the pME18S-FL3 vector (GenBank Accession No.
- telomeres for expression in cultured cells
- pMEl8S vector Mol. Cell Biol. 8: 466-472, 1988
- the insertion of a DNA of the present invention into a vector can be carried out by conventional methods, for example, by a ligase reaction using restriction enzyme sites (Current Protocols in Molecular Biology, ed. by Ausubel et al . , John Wiley & Sons, Inc. 1987, Section 11.4-11.11).
- the host cell into which the vector of the present invention is introduced is not specifically limited, and various host cells can be used according to the objectives of this invention.
- cells that can be used to express the proteins include, but are not limited to, bacterial cells (e.g., Streptococcus, Staphy loco ecus, E. coli, Streptomyces, Bacillus subtilis) , fungal cells (e.g., yeast, Aspergillus) , insect cells ⁇ e . g. , Drosophila S2, Spodoptera SF9) , animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cell), and plant cells.
- bacterial cells e.g., Streptococcus, Staphy loco ecus, E. coli, Streptomyces, Bacillus subtilis
- fungal cells e.g., yeast, Aspergillus
- the transfection of a vector to a host cell can be carried out by conventional methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, ed. by Ausubel et al . , John Wiley & Sons, Inc. 1987, Section 9.1-9.9), the Lipofectamine method (GIBCO-BRL) , and microinjection.
- conventional methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, ed. by Ausubel et al . , John Wiley & Sons, Inc. 1987, Section 9.1-9.9), the Lipofectamine method (GIBCO-BRL) , and microinjection.
- the protein expressed in host cells can be secreted into the lumen of the endoplasmic reticulum, into cavities around the cells, or into the extracellular environment. These signals may be endogenous or exogenous to the protein of interest.
- a protein of the present invention When a protein of the present invention is secreted into the culture medium, it is collected from that medium. If a protein of the present invention is produced intracellularly, the cells are lysed and then the protein is collected.
- a protein of the present invention can be collected and purified from a recombinant cell culture using methods known in the art, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anionic or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.
- isolated compounds refers to being separated from the original environment (for example, the natural environment if it is naturally-occurring) .
- substantially purified refers to a state where the compound has been separated from the original environment, and from which at least 60%, preferably 75%, and most preferably 90% of other coexisting natural components have been removed.
- the present invention provides an antimalarial drug that inhibits the activity of the GWTl gene product of malaria parasites.
- a preferred compound inhibiting the activity of the GWTl gene product of malaria parasites is the compound described in WO 02/04626, and includes the compounds (1) to (5) : 15
- a Compound that inhibits the activity of the malaria parasite GWTl gene product, or a salt thereof, or a hydrate thereof, can be administered as it is to mammals (preferably humans) . It can also be formulated by a conventional method into a tablet, powder, fine granule, granule, coated tablet, capsule, syrup, troche, inhalant, suppository, injection, ointment, eye ointment, eye drop, nasal drop, ear drop, cataplasm, lotion, and such, and then administered.
- auxiliary agents ordinarily used in pharmaceutical formulations for example, fillers, binders, lubricants, coloring agents, flavoring agents, and as necessary, stabilizers, emulsifiers, absorbefacient, surfactants, pH regulators, antiseptics, and antioxidants
- a pharmaceutical formulation can be prepared using an ordinary method combining components that are generally used as ingredients for pharmaceutical preparations.
- oral formulations can be produced by combining a compound of the present invention or a pharmaceutically acceptable salt thereof with a filler, and as necessary, a binder, disintegrator, lubricant, coloring agent, flavoring agent, and such, and then formulating the mixture into a powder, fine granule, granule, tablet, coated tablet, capsule, and such by usual methods.
- these components include: animal fat and vegetable oils such as soybean oil, beef tallow, and synthetic glyceride; hydrocarbons such as liquid paraffin, squalene, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resin; silicone oil; surfactants such as polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hardened castor oil, and polyoxyethylene polyoxypropylene block copolymer; water-soluble macromolecules such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone, and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerol
- fillers include lactose, corn starch, refined white sugar, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide.
- Binders are polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl ethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polypropyleneglycol polyoxyethylene block polymer, meglumine, and such.
- disintegrators examples include starch, agar, powdered gelatin, crystalline cellulose, calcium carbonate, sodium hydrogencarbonate, calcium citrate, dextrin, pectin, and calcium carboxymethylcellulose.
- Lubricants are magnesium stearate, talc, polyethyleneglycol, silica, hardened vegetable oil, and such.
- coloring agents are those accepted for addition to pharmaceuticals.
- Flavoring agents are cocoa powder, I-menthol, aromatic dispersant, mint oil, borneol, cinnamon powder, and such. The use of sugar coating and other appropriate coating as necessary is of course permissible for these tablets and granules.
- liquid formulations such as syrups and injections can be prepared using conventional methods.
- pH regulators, solubilizers, isotonizing agents, and such, and as necessary solubilizing adjuvants, stabilizers, and so on are added to the compounds of this invention or pharmaceutically acceptable salts thereof.
- base materials used for formulation can be selected from various materials ordinarily used for medicaments, quasi-drugs, cosmetics, and such.
- the base materials to be used are, for example, animal fat and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, water soluble macromolecules, clay minerals, and purified water.
- pH regulators, antioxidants, chelating agents, antiseptic and antifungal agents, coloring matters, fragrances, and such may also be added.
- the base materials of the external formulations of the present invention are not limited thereto.
- components such as those that have a differentiation-inducing effect, blood flow accelerants, fungicides, antiphlogistic agents, cell activators, vitamins, amino acids, humectants, and keratolytic agents can be combined.
- the above-mentioned base materials are added in an amount that leads to the concentration usually used for external formulations.
- salt as described in the present invention preferably includes, for example, a salt with an inorganic or organic acid, a salt with an inorganic or inorganic base, or a salt with an acidic or basic amino acid.
- a pharmaceutically acceptable salt is preferable. Acids and bases form salts at an appropriate ratio of 0.1 to 5 molecules of acid or base to one molecule of the compound.
- a salt with an inorganic acid are a salt with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.
- a salt with an organic acid includes a salt with acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, and p-toluenesulfonic acid.
- a salt with an inorganic base are: an alkali metal salt such as a sodium salt and a potassium salt; an alkaline earth metal salt such as a calcium salt and a magnesium salt; an aluminum salt, and an ammonium salt.
- a salt with an organic base includes a salt with diethylamine, diethanolamine, meglumine, and N,N' -dibenzylethylenediamine.
- a salt with an acidic amino acid are a salt with aspartic acid and glutamic acid, and preferably, a salt with a basic amino acid includes a salt with arginine, lysine, and ornithine.
- the compounds of the present invention or salts thereof, or hydrates thereof can be administered orally or parenterally by a conventional method without limitation as to their form. They can be formulated into dosage forms such as tablets, powders, fine granules, capsules, syrups, troches, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, cataplasms, and lotions.
- the dose of the pharmaceutical compositions of this invention can be selected appropriately depending on the degree of the symptoms, the patient's age, sex and weight, the dosage form, the type of salt, the specific type of disease, and such.
- therapeutically effective dose refers to the amount of pharmaceutical agent that yields the desired pharmacological result and is effective in the recovery or relief from the symptoms of the patient to be treated.
- the dose differs markedly depending on the type of disease, the degree of symptoms, the patient's weight, age, sex, sensitivity to the agent.
- the normal adult dosage for one day is about 0.03 mg to 1000 mg, preferably 0.1 mg to 500 mg, more preferably 0.1 mg to 100 mg, when administered from once to several times a day, or from once to several times over several days.
- the dose for injections is normally, about 1 to 3000 ⁇ g/kg, and is preferably about 3 to 1000 ⁇ g/kg.
- the present invention relates to a method of screening for antimalarial drugs using the malaria parasite GWTl gene product.
- a screening method includes, but is not limited to:
- a binding assay which screens for compounds that compete with a labeled compound to bind with the malaria parasite GWTl gene product
- a GlcN-PI acyltransferase assay system to screen for compounds that inhibit the GlcN-PI acyltransferase activity of the malaria parasite GWTl gene product; and [3] A GPI-anchored protein detection system in which the malaria parasite GWTl gene product is expressed in cells, preferably fungal cells, and then the GPI-anchored proteins on the cell surface are detected.
- the present invention is not limited to these methods, and comprises any method of screening for antimalarial drugs using the malaria parasite GWTl gene product. The methods [1] to [3] listed above are described below in detail.
- the two methods according to the present invention are disclosed below, namely (1) a method for preparing the malaria parasite GWTl gene product (hereinafter referred to as the malaria parasite GWTl protein) and (2) a method for a binding experiment involving a labeled compound (hereinafter referred to as a binding assay) .
- the malaria parasite GWTl protein is prepared from a cell membrane fraction, preferably from fungal cells, more preferably from cells of S. cerevisiae into which the DNA encoding the malaria parasite GWTl protein of SEQ ID NO: 2 has been introduced. It is preferable to introduce such a DNA into GWTl gene-deficient cells .
- the prepared membrane fraction may be used without any further treatment, or can be further purified before use. The procedure using S. cerevisiae is described below in detail, (a) Introduction of the malaria parasite GWTl gene
- the malaria parasite GWTl gene used in the present invention can be a naturally-occurring gene, or preferably, it can be synthesized based on the amino acid sequence of SEQ ID NO: 2 or 4.
- the malaria parasite GWTl gene is very rich in adenine and thymine. Thus, it was predictable that the gene will be difficult to manipulate with ordinary gene recombination techniques, and that gene expression in yeast, cells, and such will be inefficient.
- An expression plasmid for the malaria parasite GWTl is prepared by inserting the malaria parasite GWTl gene into an S. cerevisiae expression vector, for example, an expression vector prepared by inserting a suitable promoter and terminator, such as the pKTIO-derived GAPDH promotor and GAPDH terminator, into the expression vector YEp352's multi-cloning site (Tanaka et al . , Mol. Cell Biol., 10:4303-4313, 1990).
- a suitable promoter and terminator such as the pKTIO-derived GAPDH promotor and GAPDH terminator
- cerevisiae e.g., G2-10 strain
- an appropriate medium e.g., YPD medium (Yeast extract-Polypeptone-Dextrose medium)
- an appropriate temperature e.g. 30°C
- the GWTl-expression plasmid is introduced into S. cerevisiae cells using, for example, the lithium-acetate method. This method is described in the User Manual of YEAST MAKERTM Yeast Transformation System (BD Biosciences Clontech) .
- a malaria parasite GWTl-overexpressing strain and a strain carrying a negative control vector can be obtained by culturing the transformed cells on SD (ura-) medium at 30°C for two days .
- expression vectors and gene transfer methods for fungal species other than S. cerevisiae have been reported as follows: expression vectors such as pcL for Schizosaccharomyces pombe ⁇ S. pombe) and their transfer methods are described by Igarashi et al. (Nature 353:80-83, 1991) ; expression vectors such as pRMlO for C. albicans and their transfer methods are described by Pla J. etal. (Yeast, 12: 1677-1702, 1996) ; expression vectors such as pAN7-l for A. fumigatus and their transfer methods are described by Punt P. J. etal.
- S. cerevisiae cells in which the malaria parasite GWTl gene has been introduced are cultured in an appropriate medium (e.g., SD (ura-) liquidmedium) while being shaken at an appropriate temperature (e.g., 30 °C) .
- an appropriate medium e.g., SD (ura-) liquidmedium
- an appropriate temperature e.g. 30 °C
- homogenization buffer e.g., 50 mM Tris-HCl, pH7.5, 10 mM EDTA, CompleteTM (Roche)
- glass beads e.g., four times the volume of fungal cells
- the homogenization buffer is added to the resulting lysate.
- the mixture is centrifuged, for example at 2,500 rpm for five minutes, to precipitate the glass beads and uncrushed fungal cells.
- the supernatant is transferred to another tube.
- the tube is centrifuged, for example at 13,500 rpm for ten minutes, to precipitate a membrane fraction (total membrane fraction) comprising organelles.
- the precipitate is suspended in 1 ml of binding buffer (e.g., 0.1 M Phosphate buffer, pH 7.0, 0.05% Tween 20, CompleteTM (Roche)), and then centrifuged, for example, at 2,500 rpm for one minute to remove unsuspended material.
- the supernatant is then centrifuged, for example at 15,000 rpm for five minutes.
- the precipitate is resuspended in 150to 650 ⁇ l of binding buffer to prepare a membrane fraction.
- Membrane fractions can be prepared from fungal species other than S . cerevisiae using the method of Yoko-o et al . for S. pombe (Eur. J. Biochem. 257:630-637, 1998); the method of Sentandreu M et al . for C. albicans (J. Bacteriol., 180: 282-289, 1998); the method of Mouyna I et al . for A. fumigatus (J. Biol. Chem. , 275: 14882-14889, 2000); and the method of Thompson JR et al . for C. neoformans (J. Bacteriol., 181: 444-453, 1999).
- the malaria parasite GWTl protein can be prepared by expressing an E. coli, insect and mammalian cell or the like in non-fungal cells.
- the malaria parasite GWTl gene is ligated with an over-expression vector containing, for example, the CMV promotor, and then introduced into the mammalian cells.
- Membrane fractions can then be prepared according to the method of Petaja-Repo et al . (J. Biol. Chem., 276:4416-23, 2001).
- Insect cells expressing the malaria parasite GWTl gene can be prepared using, for example, a baculovirus expression kit such as the BAC-TO-BAC® Baculovirus Expression system (Invitrogen) . Membrane fractions can then be prepared according to the method of Okamoto et al . (J. Biol. Chem., 276:742-751, 2001).
- the malaria parasite GWTl protein can be prepared from E. coli by, for example, ligating the malaria parasite GWTl gene into an E. coli expression vector such as the pGEX vector (Pfizer Inc.), and introducing the construct into E. coli such as BL21.
- E. coli expression vector such as the pGEX vector (Pfizer Inc.)
- the labeled compound is prepared from a compound that has been confirmed to bind to GWTl proteins. Any compound which can bind to GWTl proteins can be used.
- the labeled compound is preferably prepared from the compound described in WO 02/04626, more preferably from compounds according to (1) to (5) described above.
- the compound is labeled with a radioisotope, more preferably with 3 H.
- the radiolabeled compound can be prepared by typical production methods using a radioactive compound as a starting material.
- 3 H labeling can be achieved using an 3 H exchange reaction.
- the labeled compound is added to the prepared membrane fraction and the mixture is allowed to stand on ice for an appropriate time, for example, one to two hours, while the binding reaction between the labeled compound and the membrane fraction takes place.
- the membrane fraction is precipitated by centrifuging the mixture, for example at 15,000 rpm for three minutes.
- the precipitate is resuspended in binding buffer, and the suspension is centrifuged. This is repeated appropriately (twice) to remove any unbound labeled compound.
- the precipitate is again suspended in binding buffer.
- the resulting suspension is transferred into a scintillation vial, and a scintillator is added. Radioactivity is measured using a liquid scintillation counter.
- the specific binding of the labeled compound to the GWTl protein can be confirmed by assessing whether binding of the labeled compound is inhibited by adding a large excess of unlabeled compound (ten times or more) , and whether the compound binds negligibly to membrane fractions prepared from fungal cells which do not express the GWTl protein.
- Test compounds used in the present invention's screening method include: a simple naturally-occurring compound, an organic compound, an inorganic compound, a protein, or a peptide, as well as a compound library, an expression product of a genetic library, a cell extract, a cell culture supernatant, a product from fermentative bacteria, an extract of a marine organism, a plant extract, and the like.
- the mixture is centrifuged, for example at 15,000 rpm for three minutes to precipitate the membrane fraction.
- the precipitate is resuspended in binding buffer and the suspension is centrifuged. This is repeated appropriately (twice) to remove any unbound labeled compound.
- the precipitate is suspended in the binding buffer.
- the suspension is transferred into a scintillation vial, and scintillator is added thereto. The radioactivity is measured using a liquid scintillation counter.
- the test sample is judged to have the activity of binding to the malaria parasite GWTl protein.
- the transfer of an acyl group to GPI can be detected by the method reported by Costello L.C and Orlean P., J. Biol. Chem. (1992) 267:8599-8603; or Franzot S.P and Doering T.L., Biochem. J. (1999) 340:25-32.
- Costello L.C and Orlean P. J. Biol. Chem. (1992) 267:8599-8603
- Franzot S.P and Doering T.L. Biochem. J. (1999) 340:25-32.
- the following experimental conditions are preferably optimized for each malaria parasite GWTl protein to be used.
- the malaria parasite GWTl protein is prepared according to the procedure described in Section 1.
- a membrane fraction comprising the malaria parasite GWTl protein is added to a buffer which comprises an appropriate metal ion (Mg 2+ , Mn 2+ ) , ATP, Coenzyme A, and preferably an inhibitor that prevents the consumption of UDP-GlcNAc in other reactions, for example, nikkomycin Z as an inhibitor of chitin synthesis, or tunicamycin as an inhibitor of asparagine-linked glycosylation.
- a test sample is then added to the mixture and the resulting mixture is incubated at an appropriate temperature for an appropriate period of time (for example, at 24 °C for 15 min) .
- a GlcN- (acyl) PI precursor for example UDP-GlcNAc, Acyl-Coenzyme A, and preferably UDP- [ 14 C] GlcNAc
- UDP-GlcNAc Acyl-Coenzyme A
- UDP- [ 14 C] GlcNAc UDP- [ 14 C] GlcNAc
- the resulting mixture is incubated for an appropriate period of time (for example, at 24 °C for one hour).
- a mixture of chloroform and methanol (1:2) is added, the resulting mixture is stirred to halt the reaction, and the lipids are extracted.
- the extracted reaction product is dissolved in an appropriate solvent, preferably butanol.
- GlcN- (acyl) PI produced in the reaction is separated by a method such as HPLC or thin layer chromatography (TLC) , preferably TLC.
- TLC thin layer chromatography
- the developer can be selected appropriately from, for example, CHC1 3 /CH 3 0H/H0 (65:25:4), CHC1 3 /CH 3 0H/1M NH 4 OH (10:10:3), and CHCl 3 /pyridine/HCOOH (35:30:7).
- a preferred developer is CHC1 3 /CH 3 0H/H 2 0 (65:25:4).
- the separated GlcN- (acyl) PI is quantified using a method appropriate for the label used. When labeled with an radioisotope, the separated GlcN- (acyl) PI can be quantified based on its radioactivity.
- the test sample is judged to have the activity of inhibiting acyl group transfer by the malaria parasite GWTl protein.
- a GPI-anchored protein detection system which comprises expressing the malaria parasite GWTl protein in cells and detecting the GPI-anchored protein on the cell surface
- the ability of a test sample to inhibit the activity of the malaria parasite GWTl protein can be determined using a GPI-anchored protein detection system that comprises expressing the GWTl protein in cells, preferably fungal cells, and then detecting the GPI-anchored protein on the cell surface.
- the fungi of the present invention are those belonging to Zygomycota, Ascomycota, Basidiomycota, and Deuteromycete, and preferably pathogenic fungi, Mucor, Saccharomyces, Candida, Cryptococcus, Trichosporon, Malassezia, Aspergillus, Trichophyton, Microsporum, Sporothrix, Blastmyces, Coccidioides, Paracoccidioides, Penicillinium, and Fusarium, more preferably C. albicans, C. glabrata, C. neoformans, and A. fumigatus, and even more preferably, yeast.
- yeasts include S. cerevisiae and S. pombe .
- the method for introducing into the above-described fungal cells an expression vector containing inserted DNA encoding the malaria parasite GWTl protein is known to those skilled in the art.
- the amount of GPI-anchored protein transported to the fungal cell wall can be determined by the following methods: (1) by using a reporter enzyme; (2) by using an antibody that reacts with the surface glycoprotein of fungal cell walls; (3) by using the protein's ability to adhere to animal cells; or (4) by observing fungal cells under a light microscope or electron microscope.
- the methods of (1) to (4) have been disclosed in WO 02/04626, which is described specifically in Examples of this invention.
- the methods (1) to (4) and preferably a combination of these methods (1) to (4) , can determine whether a test sample inhibits the transport of the GPI-anchored protein onto the cell wall, or the expression of the GPI-anchored protein on the fungal cell surface.
- the process that transports GPI-anchored proteins to the cell wall can be quantified using a tracer experiment such as one where a GPI-anchored protein is labeled with a radioactive isotope, the fungal cell wall fraction is obtained, and immunoprecipitated using an antibody against the GPI-anchored protein.
- quantification can be more readily performed as follows : the C-terminal sequence, which is considered to function as a transport signal and is commonly observed among GPI-anchored proteins, can be expressed as a fusion protein with an easily measurable enzyme (reporter enzyme) , the fungal cell wall fraction can be obtained, ; and a reporter system that measures the enzyme activity of each fraction can be used (Van Berkel MAA et al . , FEBS Letters, 349: 135-138, 1994) .
- a reporter enzyme which uses a reporter enzyme will be described, but in the present invention such methods are not to be construed as being limited thereto.
- the reporter gene is constructed and introduced into fungi.
- the reporter gene is constructed by linking a promoter sequence that functions in fungi with DNAs that respectively encode a signal sequence, a reporter enzyme, and a GPI-anchored protein C-terminal sequence in such a way that the reading frames match.
- the promoter sequence are GAL10 and ENOl.
- the signal sequence include ⁇ -factor, invertase, and lysozyme.
- reporter enzymes are ⁇ -lactamase, lysozyme, alkaline phosphatase, and ⁇ -galactosidase.
- Green Fluorescence Protein (GFP) , which has no enzyme activity but can be easily detected, can also be used.
- GPI-anchored protein C-terminal sequences include the ⁇ -agglutinin C-terminal sequence, the CWP2 C-terminal sequence, and so on. Furthermore, it is preferable to insert an appropriate selection marker, such as LEU2 and URA3, into the vector comprising the constructed reporter gene.
- the constructed reporter gene is inserted into fungi using an appropriate method, such as the lithium acetate method (Gietz D et al . , Nucl. Acids Res. 20: 1425, 1992) .
- the fungi are then cultured, as necessary, using a method that suits the selection marker (e.g. using Leu " medium for LEU2 and Ura " medium for URA3) , and then fungi into which the DNA has been introduced are selected.
- the reporter gene-introduced fungi are cultured under appropriate conditions, for example at 30 °C for 48 hours, in the presence of a test sample. After culturing, the culture supernatant is centrifuged, and the reporter enzyme activity of the culture supernatant fraction is measured. The resulting cell fraction is washed, the cell wall components are separated using an appropriate method, such as degrading the cell wall glucan with glucanase, and then the reporter enzyme activity of the cell wall fraction and cytoplasmic fraction is measured.
- the assay can be simply carried out by using centrifugation to determine the amount of reporter enzyme in the cell fraction, then without washing the cells, using proportional calculations to determine the amount of reporter enzyme derived from the culture supernatant fraction that remains in the cell fraction, and subtracting this from the amount of reporter enzyme of the cell fraction.
- test sample exhibits the activity of increasing reporter enzyme activity within the culture supernatant fraction (activity per cell) , or the activity of decreasing the reporter enzyme activity in the cell wall fraction (activity per cell) , the test sample is judged to have influenced the transport process of GPI-anchored proteins to the cell wall.
- a test sample' s ability to influence the expression of a GPI-anchored protein at the fungal surface layer can be determined by quantification using an antibody that reacts with that GPI-anchored protein in the fungal cell wall.
- Antibodies can be * obtained by predicting the antigenic determinant using the amino acid sequence of, for example, a GPI-anchored protein such as ⁇ -agglutinin, Cwp2p, or Alslp (Chen MH et al . , J. Biol. Chem., 270:26168-26177, 1995; Van Der Vaat JM et al . , J. Bacteriol., 177:3104-3110,1995; Hoyer LL et al . , Mol.
- a GPI-anchored protein such as ⁇ -agglutinin, Cwp2p, or Alslp
- a monoclonal antibody against a GPI-anchored protein may be obtained by immunizing mice and such with fungi, preferably fungi which overexpress a GPI-anchored protein such as ⁇ -agglutinin, Cwp2p, and Alslp, (in some cases by immunizing further with a partially purified GPI-anchored protein) , and then using ELISA, Western blot analysis, and so on to select resultant clones based on the antibody that they produce.
- fungi preferably fungi which overexpress a GPI-anchored protein such as ⁇ -agglutinin, Cwp2p, and Alslp
- the following method can be used to determine the influence of a test sample on the process that transports a GPI-anchored protein to the cell wall, and on the amount of protein derived from that GPI-anchored protein in the cell wall.
- Fungi are cultured in the presence of a test sample under appropriate conditions such as 30 °C for 48 hours.
- the cultured fungi are collected by centrifugation and the cells are disrupted, preferably using glass beads.
- the washed, disrupted cells are preferably subjected to centrifugal extraction with SDS, and then the precipitate is washed.
- the disrupted cells are treated with an enzyme that degrades glucan, preferably glucanase, and the centrifuged supernatant thereof is the GPI-anchored protein sample.
- the anti-Alslp peptide antibody is coated onto a 96-well plate by overnight incubation at 4°C.
- the plate is washed with a washing solution, preferably PBS comprising 0.05% Tween 20 (PBST) , and blocking is carried out using a reagent that blocks the non-specific adsorption sites of the 96-well plate, preferably a protein such as BSA or gelatin, more preferably BlockAce (Dainippon Pharmaceutical Co., Ltd.).
- the plate is again washed with a washing solution, preferably PBST, and an appropriately diluted GPI-anchored protein sample is added.
- the reaction is then carried out for an appropriate time such as two hours at room temperature.
- an antibody against the enzyme-labeled C. albicans preferably HRP-labeled anti- Candida antibody
- the labeling method may be enzyme labeling or radioactive isotope labeling.
- the amount of Alslp in the GPI-anchored protein sample is calculated by a method appropriate to the type of label, i.e. for an enzyme label, by adding a substrate solution and then, upon stopping the reaction, measuring absorbance at 490 nm.
- the test sample s influence on the expression of a GPI-anchored protein on the fungal surface can be determined by measuring the activity of that GPI-anchored protein in the fungal cell wall, and preferably by measuring the ability of fungi to adhere to animal cells and the like.
- GPI-anchored protein activity includes that of ⁇ -agglutinin in mating, of Flolp in yeast aggregation, and so on.
- a fungus with the ability to adhere to cells is used, and this fungus is preferably C. albicans .
- cells that adhere to the fungus preferably intestinal epithelial cells, are used.
- the mammalian cells are cultured and fixed using an appropriate method, such as ethanol fixation.
- the test sample and the fungi are incubated for an appropriate time such as 48 hours at 30 °C, then inoculated and cultured for a set time, for example, one hour at 30 °C.
- the culture supernatant is then removed, and the cells are washed with a buffer and overlaid with agar media such as Sabouraud Dextrose Agar Medium (Becton Dickinson Company, Ltd.). After culturing at 30 °C overnight, the number of colonies is counted, and the adhesion rate is calculated.
- test sample when compared to fungi not treated with the compound, a test sample is observed to have the activity of decreasing the number of colonies formed by cell adhesion, that test sample is judged to have influenced the process that transports GPI-anchored proteins to the cell wall.
- the influence of a test sample on the expression of the GPI-anchored protein in the fungal surface can be determined by observing the structure of the fungal cell wall using an electron microscope.
- a fungus such as C. albicans is cultured for a certain period of time, for example, 48 hours at 30 °C, and its ultrafine morphological structure is observed using a transmission electron microscope.
- observation using a transmission electron microscope can be carried out, for example by the method according to the Electron Microscope Chart Manual (Medical Publishing Center) .
- the flocculent fibrous structure of the outermost layer of a fungal cell has a high electron density and is observable by transmission electron microscope.
- This structure is not influenced by other existing antifungal agents and is considered to be a surface glycoprotein layer, including GPI-anchored proteins as its constituents. When this structure disappears, leaving only a slight layer with a high electron density, the test sample is judged to have influenced the process that transports GPI-anchored proteins to the cell wall, compared to untreated cells.
- test sample is judged to have an influence on the cell wall.
- the present invention also provides a method for treating malaria, which comprises the step of administering a compound that inhibits the activity of a GWTl protein a malaria parasite.
- a compound includes the compounds described in WO 02/04626 (for example, the compounds described herein in (l)-(5)).
- the nucleotide sequence for the natural PfGWTl protein is characterized by an exceedingly high AT content (80.41%), and thus codon usage is biased.
- the gene contains sequence stretches comprising six or more consecutive A residues at 23 separate positions, and these sequence stretches may serve as pseudo-poly (A) sites, thus producing truncated proteins. Because of the features described above, the gene was only expressed poorly in yeast, and very difficult to amplify using PCR or to replicate in E. coli . It was also difficult to determine the nucleotide sequence.
- the present inventors succeeded in expressing the PfGWTl protein with a high efficiency by using a degenerate mutant of the DNA (SEQ ID NO: 5) , with a lower AT content than the DNA encoding the PfGWTl protein.
- the inventors also revealed that the introduction of the degenerate mutant DNA can rescue the phenotype of GWTl-deficient yeast. This finding suggests that the GPI synthase of a malaria parasite is interchangeable with that of a fungus such as yeast.
- the AT content of the gene encoding the malaria parasite GPI synthase is, for example, 79.35% for GPI8 and 77.89% for the GPI13 of P. falciparum . These AT contents are as high as that of PfGWTl. It is predicted that most P. falciparum genes are hardly expressed in other species, because the average AT content over the translated regions of the P. falciparum genome is 76.3%.
- the present inventors succeeded in expressing a degenerate mutant of the DNA with a lower AT content than that of the DNA encoding the PfGWTl protein, in yeast.
- the malaria parasite GPI synthase can be expressed in a host other than malaria parasites by using such a degenerate DNA mutant.
- GPI-deficient yeast and GWTl-deficient yeast are known to exhibit similar phenotypes, including the characteristic of lethality and such.
- the phenotype of the GPI synthase gene-deficient fungus can be rescued by using the degenerate mutant DNA described above.
- the phenotype of the GPI synthase gene-deficient fungus into which the degenerate mutant DNA described above has been introduced depends on the activity of the malaria parasite GPI synthase . Accordingly, compounds that inhibit the activity of the malaria parasite GPI synthase can be selected by screening using the phenotype of the GPI synthase gene-deficient fungus as an index. Thus, antimalarial drugs targeting the GPI biosynthesis pathway can be selected without actually using the malaria parasites themselves.
- the present invention provides a degenerate mutant DNA encoding a protein that has the activity of rescuing the phenotype of a GPI synthase gene-deficient fungus, and which has an AT content lower than that of the original DNA encoding the protein involved in the biosynthesis of GPI.
- a DNA can be used in the screening method of the present invention.
- AT content refers to the content of adenine and thymine in the entire nucleotide sequence of the coding region of the GPI synthase gene.
- the AT content in the degenerate mutant DNA of the present invention preferably ranges from 50% to 70%, more preferably from 53% to 65%, and still more preferably from 55% to 62%.
- the phenotype of the GPI synthase gene-deficient fungus includes temperature sensitivity (preferably, sensitivity to high temperatures) and lethality.
- the proteins of the present invention involved in the biosynthesis of GPI in malaria parasites include GWTl, GPIl, GPI8, GPI3/PIG-A, GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-0, GAAl/GAA-1, DPMI, GPI2, GPI15, YDR437W, GPI12, MCD4, GPI11, GPI7, GPI17, GPI16, CDC91, DPM2, DPM3, and SL15.
- GPIl and GPI8 have been found to be present in malaria parasites, and GPI3/PIG-A, GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-0, GAAl/GAA-1, and DPMI have been suggested to be present in malaria parasites (Delorenzi et al . , Infect. Immun. 70: 4510-4522, 2002).
- SEQ ID NO: 1 the even sequence identification numbers in SEQ ID NOs: 6-21, respectively.
- Each corresponding amino acid sequence is shown in SEQ ID NO: 2 and the odd sequence identification numbers in SEQ ID NOs: 6-21.
- nucleotide sequence of P. vivax GWTl is shown in SEQ ID NO: 3
- the corresponding amino acid sequence is shown in SEQ ID NO: 4.
- a method using hybridization or PCR GWTl, GPIl, GPI8, GPI3/PIG-A, GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-0, GAAl/GAA-1, or DPMI of other malaria parasites can be cloned using DNA comprising any one of the nucleotide sequences shown in SEQ ID NO: 1 and 3, and the even-numbered SEQ ID NOs: 6-21.
- GPI synthase genes other than GWTl, GPIl, GPI8, GPI3/PIG-A, GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-0, GAAl/GAA-1, and DPMI of malaria parasites can be cloned by using yeast or human GPI synthase genes.
- the nucleotide sequences of GPI2, GPI15, YDR437W, GPI12, MCD4, GPIll, GPI7, GPI17, GPI16, and CDC91 of yeast S.
- each corresponding amino acid sequence is shown in the odd sequence identification numbers in SEQ ID NOs: 22-41.
- the nucleotide sequences of human DPM2, DPM3, and SL15 are shown in the even sequence identification numbers in SEQ ID NOs: 42-47 respectively; and each corresponding amino acid sequence is shown in the odd sequence identification numbers in SEQ ID NOs: 42-47.
- the production of a degenerate mutant DNA encoding a protein involved in the biosynthesis of the GPI of malaria parasites, and with a lower AT content than that of the original DNA consists of two steps: design, and synthesis.
- design step the amino acid sequence of a protein of interest is first reverse-translated and then possible codons for each amino acid residue are listed.
- Reverse translation can be achieved by using commercially available gene analysis software (for example, DNASIS-Pro; Hitachi Software Engineering Co., Ltd). Of the codons listed, those meeting the purpose (for example, codons whose AT content is lower and codons frequently used in the host to be used for gene expression) are selected for each amino acid.
- the degenerate mutant DNA can be designed by rearranging the amino acid sequence of the protein of interest using these selected codons.
- the DNA thus designed can be synthesized by a method known to those skilled in the art.
- the degenerate mutant DNA of the present invention can be synthesized based on the designed nucleotide sequence by, for example, using a commercially available DNA synthesizer.
- the present invention also provides vectors in which the above-described degenerate mutant DNA has been inserted, and transformants (preferably GPI synthase gene-deficient fungi) that retain the DNA or the vector in an expressible state.
- transformants preferably GPI synthase gene-deficient fungi
- the vector and the host can be those described above.
- the expression "deficient in the GPI synthase gene” means that the functional product of the gene is not expressed, or that the expression level is decreased.
- the GPI synthase gene-deficient fungus of the present invention can be prepared by disrupting the GPI gene. The disruption can be achieved by inserting DNA unrelated to the gene, for example a selection marker, based on homologous recombination technology, and the like. More specifically, such a mutant fungus can be prepared by introducing into yeast a selection marker cassette which comprises the his5 gene or the kanamycin resistance gene of S . pombe (Longtine et al . , Yeast, 14: 953-961, 1998) amplified with primers, each of which comprises a nucleotide sequence homologous to a portion of the gene (ranging from 50 to 70 nucleotides) .
- the GPI synthase gene-deficient fungus of the present invention includes, for example, the GWTl temperature-sensitive mutant strain gwtl-20, GPI7 disruptant strain, GPI8 mutant strain gpi8-l, and GPI10 temperature-sensitive mutant strain perl3-l.
- a GPI synthase gene-deficient fungus which has been transformed with the degenerate mutant DNA of the present invention can be prepared by introducing into a fungus a vector into which the degenerate mutant DNA has been inserted.
- pRS316, YEp351, or such can be used as the vector for S . cerevisiae, and pcL, pALSK, or such can be used as the vector for S. pombe .
- the present invention also provides a method of screening for antimalarial drugs, which comprises using GPI synthase gene-deficient fungi described above.
- the first step comprises contacting a test sample with a GPI synthase gene-deficient fungus that has been transformed with degenerate mutant DNA with a lower AT content than the DNA encoding a protein involved in the biosynthesis of GPI of malaria parasites.
- the "contact" can be achieved by adding a test sample to the culture of the above-mentioned fungus.
- the test sample is a protein
- a vector comprising DNA encoding the protein can be introduced into the above-mentioned fungus .
- the next step comprises measuring the degree of growth of the above-mentioned fungus. More specifically, the fungus is inoculated under typical culture conditions, specifically, the fungus is inoculated onto a liquid culture medium such as Yeast extract-polypeptone-dextrose medium (YPD medium) or onto an agar plate, and then incubated at 25 to 37 °C for 4 to 72 hours.
- a liquid culture medium such as Yeast extract-polypeptone-dextrose medium (YPD medium) or onto an agar plate
- YPD medium Yeast extract-polypeptone-dextrose medium
- the degree of growth can also be determined using the turbidity of the culture liquid, the number of colonies, or the size or color of the spots formed on the agar plate as an index.
- the next step comprises selecting compounds that inhibit the growth of the above-mentioned f ngus .
- the first step comprises contacting a test sample with a GPI synthase gene-deficient fungus in which the above-described degenerate mutant DNA has been introduced.
- the next step comprises determining the amount of GPI-anchored protein transported onto the yeast cell wall.
- the detection method includes: (1) methods using a reporter enzyme; (2) methods using an antibody that reacts with a surface glycoprotein on the fungal cell wall; (3) methods using the ability to adhere to animal cells; and (4) methods using a light microscope or an electron microscope to observe the fungi .
- the next step comprises selecting a sample that decreases the amount of GPI-anchored protein transported to the cell wall.
- the present invention provides a method of screening for antimalarial drugs using a protein involved in the biosynthesis of GPI, which is prepared using a degenerate mutant DNA of the present invention.
- Such methods include, for example, a binding assay system where screening is carried out to select compounds that bind to a protein involved in GPI biosynthesis in competition with a labeled compound bound to the protein.
- a degenerate mutant DNA of the present invention is introduced into the GPI synthase gene-deficient fungus, the protein encoded by the DNA is expressed in the fungus, and the expressed protein is prepared.
- the prepared protein is then contacted with a test sample and with a labeled compound that can bind to the protein.
- the labeled compound bound to the protein is detected, and test samples that decrease the amount of labeled compound bound to the protein are selected.
- the present invention also provides an assay system for GlcN-PI acyltransferase.
- a system comprises using a GWTl protein which is prepared using a DNA encoding a protein that has the activity of complementing the phenotype of GWTl-deficient yeast, which the DNA is a degenerate mutant of a DNA encoding a malaria parasite GWTl protein that has a lower AT content than the original DNA.
- the degenerate mutant DNA is introduced into GWTl-deficient fungus, the protein encoded by the degenerate mutant DNA is expressed in the fungus, and the expressed protein is prepared.
- This protein is then contacted with a test sample, GlcN- (acyl) PI is detected, and a test sample that decreases the amount of GlcN- (acyl) PI is selected.
- Fig. 1 depicts photographs showing the results of tetrad analysis.
- the gwtl-disrupted strain became viable after the introduction of the opfGWTl-overexpressing plasmid.
- the four spores derived from a single diploid cell were spotted vertically.
- Fig. 2 depicts a diagram showing the inhibitory activity of a compound with respect to the growth of yeast expressing the opfGWTl gene. Either the yeast GWTl gene or opfGWTl gene was expressed in GWTl gene-disrupted yeast.
- a compound having the activity of inhibiting the GWTl-dependent growth of yeast also showed inhibitory activity with respect to the opfGWTl-dependent growth of yeast in which opfGWTl was expressed.
- Fig 3 depicts a diagram showing antimalarial activity. Human red blood cells were infected with P. falciparum . A GWTl-inhibiting compound was added to these red blood cells, and inhibition of malaria parasite infection was determined.
- PfGWTl P. falciparum GWTl
- SEQ ID NO: 1 The nucleotide sequence of P. falciparum GWTl (SEQ ID NO: 1) has been disclosed in the database of the P. falciparum genome (PlasmoDB database, http://plasmodb.org/).
- the PfGWTl gene was cloned by PCR using genomic DNA purified from P. falciparum (the 3D7 strain) as a template.
- the 5' half and 3' half of the PfGWTl gene were prepared separately, and the two halves were assembled at an Xbal (TCTAGA) restriction enzyme site.
- TCTAGA Xbal restriction enzyme site
- the 5' half of the PfGWTl gene was amplified by PCR using P. falciparum genomic DNA as a template and the primers pfl52F (SEQ ID NO: 48) and pfl36R (SEQ ID NO: 49) .
- the 3' half was amplified by the same procedure described above, using the primers pfl37F (SEQ ID NO: 50) and pfl5lR (SEQ ID NO: 51) .
- the DNA fragments amplified were subcloned into the pT7-Blue vector (Novagen) , and the nucleotide sequences of the inserts were sequenced to confirm homology to SEQ ID NO: 1.
- Clones containing the 5' half of the PfGWTl gene were named PF15-5 clones.
- Clones containing the 3' half were named PF20-9 clones .
- the 3' half was amplified by PCR using PF20-9 as a template and the primers pfl68BK (SEQ ID NO: 54) and pfl55RK (SEQ ID NO: 55) .
- the amplified DNA fragments were subcloned to prepare pT7-plasmBK5 clones.
- the full-length PfGWTl gene was prepared by the procedure described below.
- the yeast expression vector YEp352GAPII was digested with the restriction enzymes EcoRI and Kpnl.
- the EcoRI-Xbal fragment (about 1500 bp) derived from pT7-plasmN2, and the Xbal-Kpnl fragment (about 1100 bp) derived from pT7-plasmBK5, were inserted into the vector at a cleaved site.
- the expression vector YEp352GAPII-PfGWTl containing the full-length PfGWTl was then constructed.
- the adenine and thymine (AT) proportion is exceedingly high (80% or higher) in P. falciparum DNA, and thus routine biological techniques (PCR, --.. coli-based gene engineering, expression systems for recombinant proteins, and so on) are often unavailable (Sato and Horii; Protein, Nucleic acid, and Enzyme Vol. 48, 149-155, 2003) .
- the AT content of PfGWTl DNA was 80.41% including many consecutive A or T stretches.
- the gene was predicted to be difficult to replicate and express as a protein in yeast.
- the codon substitution was carried out based on the nucleotide sequence of P. falciparum GWTl (SEQ ID NO: 1) disclosed in the P. falciparum genome database (PlasmoDB database, http://plasmodb.org/) .
- the resulting nucleotide sequence was named "optimized PfGWTl (opfGWTl)" (SEQ ID NO: 5).
- the sequence described above was designed to include additional sequences outside the coding region; namely an EcoRI cleavage site sequence (GAATTC, at the 5' end) , Kozak' s sequence (ACC, at the 5' end) , and a Kpnl cleavage site sequence (GGTACC, at the 3' end) .
- the synthesis of the resulting sequence was consigned to Blue Heron Inc. in the U.S.A.
- These additional restriction enzyme sites were used to ligate the fully synthetic opfGWTl into the YEp352GAPII vector to construct an overexpression plasmid for opfGWTl.
- the construct was introduced into diploid cells (WDG2) in which only a single copy of the GWTl gene had been disrupted.
- WDG2 diploid cells
- the resulting transformants were cultured on plates containing a sporulation medium to form spores for tetrad analysis.
- the AT content of the newly designed codon-modified opfGWTl was reduced to 61.55%.
- the results of tetrad analysis are shown in Fig. 1.
- the gwtl-disrupted strain became viable after introduction of the opfGWTl overexpression plasmid.
- the findings described above indicate that the PfGWTl gene can be expressed in yeast cells when its AT content is reduced by codon modification.
- a screening system for compounds having antimalarial activity was constructed using opfGWTl-expressing yeast.
- An expression cassette was constructed by inserting the S . cerevisiae GWTl terminator, and the S. cerevisiae GAPDH promoter and multi-cloning site into the Sacl-Kpnl site of the single-copy vector pRS316.
- S. cerevisiae GWTl and opfGWTl were inserted into the multi-cloning site to prepare pGAP-ScGWTl and pGAP-opfGWTl plasmids, respectively. These plasmids were introduced into the GWTl disruptant.
- Serial two-fold dilutions of compound (1) were prepared using YPAD to make the highest final concentration 50 ⁇ g/ml.
- a 50 ⁇ l aliquot of the diluted compound was added to each well of a 96-well plate. Overnight cultures of yeast cells comprising each plasmid were diluted 1000-fold and then a 50 ⁇ l aliquot of the dilution was added to each well. The plates were incubated at 30 °C for two days, and then culture turbidity was determined at 660 nm (Fig.2 and Table 1).
- the strain became viable after introduction of each plasmid (as shown at 0 ⁇ g/ml of compound concentration) .
- the growth of ScGWTl-expressing yeast was inhibited by compound (1) , a GWTl-specific inhibitor.
- the use of the compound at 25 ⁇ g/ml resulted in about 85% inhibition of growth.
- the yeast was completely nonviable.
- the growth of opfGWTl-expressing yeast was also inhibited by compound (1) .
- the use of the compound at 25 ⁇ g/ml resulted in about 50% inhibition of growth.
- the yeast was completely nonviable.
- a test compound was dissolved in 100% DMSO, diluted with a medium, and an 80 ⁇ l aliquot of the dilution was added to each well of a 96-well culture plate.
- P. falciparum FCR3 strain was pre-cultured in RPMI1640 medium containing 10% human serum at 37 °C, and then 20 ⁇ l of the cultured cells (containing 10% red blood cells) was added to each well. At this time, 0.47% of red blood cells were infected. After culturing under 5% 0 2 , 5% C0 2 , and 90% N 2 at 37 °C for 48 hours, the malaria parasites were stained using Giemsa staining.
- the number of protozoan-infected red blood cells was determined in order to estimate infection rate (Fig 3) .
- compound (3) was revealed to have strong antimalarial activity.
- the other four compounds also showed antimalarial activity.
- Compound (4) exhibited the lowest activity. Therefore, compounds inhibiting yeast GWTl include compounds which have the activity of inhibiting P. falciparum GWTl, suggesting that antimalarial drugs can be synthesized based on such compounds .
- the present invention succeeded in producing fungi that express malaria parasite GWTl. Using such fungi, antimalarial drugs targeting the pathway of GPI biosynthesis can be screened without using malaria parasites.
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US42858902P | 2002-11-22 | 2002-11-22 | |
US428589P | 2002-11-22 | ||
PCT/JP2003/014920 WO2004048567A2 (en) | 2002-11-22 | 2003-11-21 | Methods of screening for compounds that inhibit the biosynthesis of gpi in malaria parasites |
Publications (3)
Publication Number | Publication Date |
---|---|
EP1565749A2 true EP1565749A2 (en) | 2005-08-24 |
EP1565749B1 EP1565749B1 (en) | 2011-09-21 |
EP1565749B9 EP1565749B9 (en) | 2012-04-25 |
Family
ID=32393427
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP03774152A Expired - Lifetime EP1565749B9 (en) | 2002-11-22 | 2003-11-21 | Methods of screening for compounds that inhibit the biosynthesis of gpi in malaria parasites |
Country Status (9)
Country | Link |
---|---|
US (2) | US7928215B2 (en) |
EP (1) | EP1565749B9 (en) |
JP (3) | JP4315907B2 (en) |
KR (4) | KR100933759B1 (en) |
CN (3) | CN101092627B (en) |
AT (1) | ATE525654T1 (en) |
AU (1) | AU2003282393B2 (en) |
CA (3) | CA2505067C (en) |
WO (1) | WO2004048567A2 (en) |
Families Citing this family (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ548265A (en) | 2000-07-07 | 2008-06-30 | Eisai R&D Man Co Ltd | Antifungal compounds and agents able to inhibit transport of the GPI-anchored protein to the cell wall |
ATE365224T1 (en) | 2002-11-22 | 2007-07-15 | Eisai R&D Man Co Ltd | METHOD FOR SCREENING FOR COMPOUNDS THAT INHIBIT THE ENZYMATIC ACTIVITY OF THE GWT1 GENE PRODUCT |
US7932272B2 (en) | 2003-09-30 | 2011-04-26 | Eisai R&D Management Co., Ltd. | Antifungal agent containing heterocyclic compound |
EP1782811A4 (en) | 2004-08-09 | 2010-09-01 | Eisai R&D Man Co Ltd | Novel antimalaria agent containing heterocyclic compound |
EP1864980A4 (en) | 2005-03-30 | 2010-08-18 | Eisai R&D Man Co Ltd | Antifungal agent containing pyridine derivative |
TWI385169B (en) | 2005-10-31 | 2013-02-11 | Eisai R&D Man Co Ltd | Heterocyclic substituted pyridine derivatives and antifungal agent containing same |
WO2008035726A1 (en) | 2006-09-21 | 2008-03-27 | Eisai R & D Management Co., Ltd. | Pyridine derivative substituted by heteroaryl ring, and antifungal agent comprising the same |
TW200841879A (en) | 2007-04-27 | 2008-11-01 | Eisai R&D Man Co Ltd | Pyridine derivatives substituted by heterocyclic ring and phosphonoamino group, and anti-fungal agent containing same |
JP5354542B2 (en) * | 2007-09-14 | 2013-11-27 | 国立大学法人 長崎大学 | P. falciparum vaccine candidate molecule |
US8513287B2 (en) | 2007-12-27 | 2013-08-20 | Eisai R&D Management Co., Ltd. | Heterocyclic ring and phosphonoxymethyl group substituted pyridine derivatives and antifungal agent containing same |
EP2282769A4 (en) | 2008-04-29 | 2012-04-25 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
JP2011523853A (en) | 2008-06-03 | 2011-08-25 | アボット・ラボラトリーズ | Dual variable domain immunoglobulins and uses thereof |
US8188119B2 (en) | 2008-10-24 | 2012-05-29 | Eisai R&D Management Co., Ltd | Pyridine derivatives substituted with heterocyclic ring and γ-glutamylamino group, and antifungal agents containing same |
UY33492A (en) | 2010-07-09 | 2012-01-31 | Abbott Lab | IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME |
MX341579B (en) | 2010-08-03 | 2016-08-25 | Abbvie Inc * | Dual variable domain immunoglobulins and uses thereof. |
WO2013192517A2 (en) * | 2012-06-21 | 2013-12-27 | Whitehead Institute For Biomedical Research | Compounds for treating infectious diseases |
KR102063576B1 (en) * | 2012-07-24 | 2020-01-10 | 한국전자통신연구원 | Method and apparatus for providing concealed software execution environment based on virtualization |
KR101954733B1 (en) * | 2012-10-26 | 2019-03-06 | 삼성전자주식회사 | System-on-chip processing secured contents and mobile device comprising the same |
CN109825514A (en) * | 2019-01-24 | 2019-05-31 | 华中农业大学 | Albumen and the application of vole Babesia GPI10 gene and its coding |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19640817A1 (en) | 1996-10-02 | 1998-05-14 | Hermann Prof Dr Bujard | Recombinant manufacturing process for a complete malaria antigen gp190 / MSP 1 |
WO1999021986A1 (en) * | 1997-10-29 | 1999-05-06 | Shanghai Second Medical University | Cbfbga09: a human sl15 homolog |
AU2392300A (en) | 1998-12-31 | 2000-07-31 | Millennium Pharmaceuticals, Inc. | Use of essential (saccharomyces) genes and polypeptides |
WO2001013923A1 (en) * | 1999-08-20 | 2001-03-01 | Georgetown University | Malaria gpi anchors as vaccines anti-parasitic drugs and for use in diagnostics |
US20040038239A1 (en) * | 2000-07-07 | 2004-02-26 | Kappei Tsukahara | Fungal cell wall synthesis gene |
NZ548265A (en) * | 2000-07-07 | 2008-06-30 | Eisai R&D Man Co Ltd | Antifungal compounds and agents able to inhibit transport of the GPI-anchored protein to the cell wall |
AU2002225533A1 (en) | 2000-12-22 | 2002-07-08 | Stichting Biomedical Primate Research Centre | Efficient expression of plasmodium apical membrane antigen 1 in yeast cells |
DE10254397B4 (en) | 2002-11-21 | 2010-09-23 | Poloplast Gmbh & Co. Kg | Universal hose coupling |
ATE365224T1 (en) | 2002-11-22 | 2007-07-15 | Eisai R&D Man Co Ltd | METHOD FOR SCREENING FOR COMPOUNDS THAT INHIBIT THE ENZYMATIC ACTIVITY OF THE GWT1 GENE PRODUCT |
-
2003
- 2003-11-21 KR KR1020067027915A patent/KR100933759B1/en not_active IP Right Cessation
- 2003-11-21 AU AU2003282393A patent/AU2003282393B2/en not_active Ceased
- 2003-11-21 KR KR1020077012681A patent/KR20070065452A/en not_active Application Discontinuation
- 2003-11-21 US US10/535,928 patent/US7928215B2/en not_active Expired - Fee Related
- 2003-11-21 KR KR1020107004569A patent/KR20100039428A/en not_active Application Discontinuation
- 2003-11-21 WO PCT/JP2003/014920 patent/WO2004048567A2/en active IP Right Grant
- 2003-11-21 KR KR1020057009212A patent/KR100825547B1/en not_active IP Right Cessation
- 2003-11-21 AT AT03774152T patent/ATE525654T1/en not_active IP Right Cessation
- 2003-11-21 CA CA2505067A patent/CA2505067C/en not_active Expired - Fee Related
- 2003-11-21 CN CN2007101042868A patent/CN101092627B/en not_active Expired - Fee Related
- 2003-11-21 EP EP03774152A patent/EP1565749B9/en not_active Expired - Lifetime
- 2003-11-21 JP JP2004555010A patent/JP4315907B2/en not_active Expired - Fee Related
- 2003-11-21 CA CA2721200A patent/CA2721200A1/en not_active Abandoned
- 2003-11-21 CA CA2704070A patent/CA2704070A1/en not_active Abandoned
- 2003-11-21 CN CNB2003801091050A patent/CN100504392C/en not_active Expired - Fee Related
- 2003-11-21 CN CN2007101042872A patent/CN101092628B/en not_active Expired - Fee Related
-
2008
- 2008-09-22 JP JP2008242395A patent/JP2009077715A/en active Pending
-
2009
- 2009-03-24 JP JP2009071722A patent/JP4376297B2/en not_active Expired - Fee Related
-
2011
- 2011-03-04 US US13/040,970 patent/US8252294B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
See references of WO2004048567A2 * |
Also Published As
Publication number | Publication date |
---|---|
KR20100039428A (en) | 2010-04-15 |
JP4376297B2 (en) | 2009-12-02 |
CA2505067C (en) | 2011-08-02 |
JP4315907B2 (en) | 2009-08-19 |
KR20070065452A (en) | 2007-06-22 |
ATE525654T1 (en) | 2011-10-15 |
AU2003282393B2 (en) | 2007-07-05 |
JP2009077715A (en) | 2009-04-16 |
KR20050085105A (en) | 2005-08-29 |
KR100825547B1 (en) | 2008-04-25 |
WO2004048567A3 (en) | 2004-10-14 |
KR20070010088A (en) | 2007-01-19 |
JP2009183290A (en) | 2009-08-20 |
US20060172404A1 (en) | 2006-08-03 |
WO2004048567A2 (en) | 2004-06-10 |
EP1565749B1 (en) | 2011-09-21 |
CA2505067A1 (en) | 2004-06-10 |
US8252294B2 (en) | 2012-08-28 |
US7928215B2 (en) | 2011-04-19 |
CN101092628B (en) | 2010-11-03 |
CN100504392C (en) | 2009-06-24 |
US20110201816A1 (en) | 2011-08-18 |
CN101092627A (en) | 2007-12-26 |
JP2006506996A (en) | 2006-03-02 |
CN1742203A (en) | 2006-03-01 |
CN101092628A (en) | 2007-12-26 |
AU2003282393A1 (en) | 2004-06-18 |
CN101092627B (en) | 2011-08-17 |
CA2704070A1 (en) | 2004-06-10 |
CA2721200A1 (en) | 2004-06-10 |
EP1565749B9 (en) | 2012-04-25 |
KR100933759B1 (en) | 2009-12-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8252294B2 (en) | Methods of screening for compounds that inhibit the biosynthesis of GPI in malaria parasites | |
Fillinger et al. | cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans | |
Mouyna et al. | Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability | |
Watanabe et al. | E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis | |
Komachi et al. | gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O‐glycan in A spergillus nidulans and A spergillus fumigatus | |
Ichinomiya et al. | Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans | |
Polizotto et al. | Calcineurin-dependent nuclear import of the transcription factor Crz1p requires Nmd5p | |
García-Muse et al. | Characterization of B-type cyclins in the smut fungus Ustilago maydis: roles in morphogenesis and pathogenicity | |
Drgonová et al. | The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell | |
CA2251593A1 (en) | Assays and reagents for identifying anti-fungal agents, and uses related thereto | |
JP2005507636A (en) | BAX responsive genes for drug target identification in yeast and fungi | |
Bandini et al. | The nucleocytosolic O-fucosyltransferase Spindly affects protein expression and virulence in Toxoplasma gondii | |
Pérez-Sánchez et al. | Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay | |
AU2007216681B2 (en) | Methods of screening for compounds that inhibit the biosynthesis of GPI in malaria parasites | |
US20080152641A1 (en) | Inhibitors of Siderophore Biosynthesis in Fungi | |
Parrish et al. | PtdIns (3) P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway | |
Guo et al. | A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in parasitic Leishmania major | |
WO2003074728A2 (en) | Screening for modulators of pkng activity | |
Ost | Pathogen-Specific Adaptations to Conserved Signaling Pathways in Cryptococcus neoformans | |
WO2004048604A1 (en) | Method of screening compound inhibiting enzymatic activity of gwt1 gene product | |
Kriangkripipat | Characterization of protein O-mannosyltransferases and their target proteins in Aspergillus nidulans |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20050617 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR |
|
AX | Request for extension of the european patent |
Extension state: AL LT LV MK |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1076509 Country of ref document: HK |
|
17Q | First examination report despatched |
Effective date: 20061211 |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: EISAI R&D MANAGEMENT CO., LTD. |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR |
|
AX | Request for extension of the european patent |
Extension state: AL LT LV MK |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: NV Representative=s name: VOSSIUS & PARTNER |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 60338469 Country of ref document: DE Effective date: 20111208 |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: VDEP Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
LTIE | Lt: invalidation of european patent or patent extension |
Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: SI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20111222 |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 525654 Country of ref document: AT Kind code of ref document: T Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: BE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20120123 Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: RO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: IT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 Ref country code: NL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MC Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20111130 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1076509 Country of ref document: HK |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
26N | No opposition filed |
Effective date: 20120622 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: MM4A |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 60338469 Country of ref document: DE Effective date: 20120622 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20111121 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20121130 Year of fee payment: 10 Ref country code: DE Payment date: 20121121 Year of fee payment: 10 Ref country code: CH Payment date: 20121122 Year of fee payment: 10 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20121120 Year of fee payment: 10 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: ES Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20120101 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20111121 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: BG Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20111221 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20110921 |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
GBPC | Gb: european patent ceased through non-payment of renewal fee |
Effective date: 20131121 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CH Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20131130 Ref country code: LI Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20131130 |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: ST Effective date: 20140731 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R119 Ref document number: 60338469 Country of ref document: DE Effective date: 20140603 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20140603 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20131202 Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20131121 |